CN112011529B - Creatine amidino hydrolase mutant with improved activity - Google Patents
Creatine amidino hydrolase mutant with improved activity Download PDFInfo
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Abstract
The invention discloses a creatine amidinohydrolase mutant with improved activity, belonging to the technical field of enzyme engineering. The creatine amidinohydrolase sequence is analyzed by a consensus method without phylogenetic prejudice, single-point mutants with remarkably improved activity are obtained through screening and site-specific mutagenesis is carried out on the single-point mutants, and the activity of the mutant enzymes D17V/K351E, T199S/K351E, D17V/T199S/K351E and D17V/T117P/K351E with improved activity is improved by about 2 times compared with that of wild type.
Description
Technical Field
The invention belongs to the field of bioengineering, and particularly relates to a creatine amidinohydrolase mutant with improved activity.
Background
Creatine amidinohydrolase is an essential enzyme for the enzymatic detection of creatinine content, and it converts creatine into sarcosine and urea, further generating hydrogen peroxide which can be chemically detected. The enzyme is mainly derived from microorganisms, and is widely applied to industries such as medical diagnosis, organic synthesis and the like at present.
Creatine amidinohydrolase is used in industrial determination of creatinine content and, in addition, is often used in clinical analysis for diagnosis of creatinine content in serum and urine and kidney diseases different from creatinine content in healthy organisms. Creatinine is a final product of creatine phosphate metabolism applied to human body, and enters urine from blood after being filtered by kidney, and is discharged out of body. Generally, serum creatinine normally ranges between 35 and 150 μm, but when kidney function or muscle function is compromised, creatinine levels rise to 1000 μm and creatinine levels in blood and urine reflect renal excretion. The most common methods for measuring creatinine content so far are Jaffe chemical detection and enzymatic colorimetric methods. In contrast, enzymatic assays are gaining attention due to their high sensitivity and selectivity. In the enzymatic detection method, a sample to be detected is continuously converted by virtue of creatinine hydrolase, creatine amidinohydrolase and sarcosine oxidase, finally creatinine is degraded into hydrogen peroxide, and the concentration of the hydrogen peroxide is determined by virtue of a colorimetric reaction under the catalysis of horseradish peroxidase, so that the aim of detecting the content of the creatinine is fulfilled.
Therefore, in order to better apply the creatine amidino hydrolase to clinical creatinine detection, the invention adopts site-specific mutagenesis to obtain the mutant enzyme with obviously improved activity, solves the problem that the existing creatine amidino hydrolase has poor activity and cannot meet the requirement of being applied to reagents, and lays a foundation for widening industrial application of the creatine amidino hydrolase.
Disclosure of Invention
In order to better apply the creatine amidino hydrolase to clinical creatinine detection, the invention adopts site-specific mutagenesis to obtain the mutant enzyme with obviously improved activity, solves the problem that the existing creatine amidino hydrolase has poor activity and cannot meet the requirement of being applied to a reagent, and lays a foundation for widening the industrial application of the creatine amidino hydrolase.
The first purpose of the invention is to provide a creatine amidinohydrolase mutant, which is (a 1) or (a 2) as follows:
(a1) A derived protein which is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO.1 and has the same function with the protein shown in SEQ ID NO. 1;
(a2) A derivative protein which is obtained by substituting one or more amino acid residues for one or more positions in the amino acid sequence shown in SEQ ID NO.1 and shows at least 92% homology with the protein shown in SEQ ID NO. 1.
Preferably, the creatine amidino hydrolase mutant has a mutation site of the amino acid sequence shown in SEQ ID NO.1, which comprises at least one of the following: 17 th, 58 th, 117 th, 199 th and 351 th bits.
Further preferably, the creatine amidinohydrolase mutant comprises a single point mutant of any one of single point mutation sites D17V, G58D, T117P, T199S and K351E in the amino acid sequence shown in SEQ ID NO. 1.
Further preferably, the creatine amidinohydrolase mutant comprises D17V/G58D, D17V/T117P, D17V/K351E, D17V/T199S, T199S/K351E, D17V/T117P/T199S, D17V/T117P/K351E at the amino acid sequence shown in SEQ ID NO. 1.
It is a second object of the present invention to provide a gene encoding a creatine amidinohydrolase mutant.
In one embodiment of the invention, the gene comprises the nucleotide sequence shown in SEQ ID NO. 2.
The third purpose of the invention is to provide a vector containing the gene.
It is a fourth object of the invention to provide cells expressing said mutant.
In one embodiment of the invention, the cell comprises a fungal cell or a bacterial cell.
In one embodiment of the invention, the cell comprises Escherichia coli, yeast or Bacillus subtilis.
It is a fifth object of the present invention to provide a method for increasing creatine amidino hydrolase activity, comprising the steps of:
1. searching the amino acid sequence of SEQ ID NO.1 in an NCBI database, deleting the repeated identical sequence, and selecting the amino acid sequence with the amino acid sequence consistency of more than 50 percent with the amino acid sequence of SEQ ID NO. 1;
2. then, performing multi-sequence comparison through ClustalX2.1 software, arranging the residual amino acid sequences into fasta files, introducing the fasta files into MEGA7.0 software, and constructing a Phylogenetic tree by utilizing an NJ algorithm in a Phylogenetic module of the MEGA7.0 software;
3. and (3) introducing weight according to the branch distance of the phylogenetic tree, calculating consensus sequence through a python script, and screening mutation sites related to activity into D17V, G58D, T117P, T199S and K351E by combining a homologous modeling structure.
The wild-type creatine amidino hydrolase has GenBank accession No. BAA88830.1, and amino acids 6, 17, 20, 52, 58, 73, 108, 166, 351, 33, 59, 109, 162, 117, 165, 199, 251, 349, 362, 340 and 331 are mutated.
The technical scheme of the invention has the following advantages:
1. compared with wild-type creatine amidino hydrolase (BAA 88830.1), the single-point mutant and the combined mutant have improved activities at 55 ℃ and 57 ℃, and the optimal activity of the mutant is about 1.6 times that of the wild-type creatine amidino hydrolase (BAA 88830.1). Based on the above, the creatine amidino hydrolase mutant provided by the invention has better activity, and the creatine amidino hydrolase mutant obtained by the construction method provided by the invention has excellent catalytic activity when catalyzing creatine to generate sarcosine and urea at higher temperature.
2. The constructed gene engineering bacteria of the creatine amidinohydrolase (BAA 88830.1) can efficiently express creatine amidinohydrolase mutants, and have the advantages of simple culture conditions, short culture period, convenient purification of expressed products and the like.
Detailed Description
Mutant naming mode:
"amino acid substituted for the original amino acid position" is used to indicate the mutant. As in G58D, the amino acid at position 58 is replaced by Glu of the parent creatine amidinohydrolase to Asp, the numbering of the positions corresponding to the amino acid sequence of the parent creatine amidinohydrolase.
Example 1: construction of single-site creatine amidinohydrolase (BAA 88830.1) mutant
Wild-type creatine amidino hydrolase plasmid Pany1-CR-AF-WT was deposited in the laboratory, and single-site creatine amidino hydrolase mutants were constructed by the whole plasmid PCR method. The details are as follows: using Pany1-CR-AF-WT as a template, the primers upstream and downstream of each mutation site are listed in Table 1, and are named in the format of "substitution of amino acid at mutation site". One round of PCR amplification was performed using the high fidelity DNA Polymerase PrimeSTAR HS DNA Polymerase kit in order to obtain a mutant-containing gene recombinant plasmid. The reaction system is shown in Table 2, and the PCR conditions are as follows: pre-denaturation: 4min at 95 ℃; denaturation: 10s at 98 ℃; and (3) annealing: 5s at 55 ℃; extension: 6min at 72 ℃; circulating for 25 times; fully extending: 10min at 72 ℃.
TABLE 2 primer Table
One round of PCR amplification was performed using high fidelity DNA Polymerase PrimeSTAR HS DNA Polymerase kit to obtain a recombinant plasmid containing the mutant. The reaction system is shown in Table 2, and the PCR conditions are as follows: pre-denaturation: 4min at 95 ℃; denaturation: 10s at 98 ℃; annealing: 5s at 55 ℃; extension: 6min at 72 ℃; circulating for 25 times; fully extending: 10min at 72 ℃.
TABLE 2 reaction System for the first round of PCR amplification
Example 2: construction of multipoint creatine amidino hydrolase (BAA 88830.1) mutant
To further analyze the effect of different amino acid species at each site on the catalytic properties of the enzyme, the whole plasmid PCR technique was still used to obtain saturated mutant library genes, with reference to the site-directed mutagenesis method, as follows: PCR amplification was performed in multiple rounds using the high fidelity DNA Polymerase PrimeSTAR HS DNA Polymerase kit in order to obtain mutant-containing recombinant plasmids. The reaction system, PCR conditions and transformation conditions were the same as those for site-directed mutagenesis.
Example 3: construction of mutant engineering bacteria
The engineering bacteria are constructed by referring to the super competence kit instruction and slightly modifying, and the specific operation is as follows. First, it was confirmed that e.coli BL21 (DE 3) could not grow under Kan resistance; secondly, scribing, separating and activating the E.coli BL21 (DE 3); thirdly, taking a single colony, adding the single colony into an LB culture medium without resistance, and culturing the single colony to OD 600 Preparing competent cells from the solution of the kit between 0.5 and 0.6; fourthly, transforming and smearing the strain on an LB solid medium plate containing Kan resistance, and culturing for 14h; and finally, selecting 5 single colonies, carrying out PCR amplification on target genes by using a bacterial solution, identifying target bands by agarose gel electrophoresis, selecting Suzhou Jinwei Zhi sequencing, and confirming engineering bacteria.
Example 4: expression and purification of creatine amidine hydrolase mutant (BAA 88830.1) protein
Inoculating the engineering bacteria in the glycerin pipe into a 4mL 2YT liquid culture medium test tube containing 100 mug/mL kanamycin (Kan +) according to the volume ratio of 1%, and culturing for 11h at 37 ℃ and 220 rpm; then transferring the 4mL bacterial liquid to a 1L shake flask containing a 2YT liquid culture medium containing 50 ug/mL kanamycin (Kan +), and culturing at 37 ℃ and 220rpm for about 3h to make OD600 reach about 0.8; 0.1mM IPTG inducer was added thereto, and the mixture was subjected to induction culture at 25 ℃ and 200rpm for 11 to 17 hours, in this example for 14 hours. And (3) centrifuging the escherichia coli thallus suspension obtained after the induction expression, and performing one-step Ni-NTA affinity chromatography treatment to obtain the creatine amidino hydrolase protein with the purity of more than 95%.
Example 5: enzyme activity determination of creatine amidino hydrolase mutant
The activity of the optimized wild-type creatine amidino hydrolase (BAA 88830.1) and various creatine amidino hydrolase mutants provided by the embodiment 3 is tested, and the method for measuring the activity of the creatine amidino hydrolase specifically comprises the following steps:
the activity detecting reaction of creatine amidinohydrolase is based on enzyme coupling catalytic system, which catalyzes creatine to generate sarcosine and urea in the reaction systemThe amino acid can react under the catalysis of Sarcosine Oxidase (SOX) and can generate hydrogen peroxide (H) 2 O 2 ) Hydrogen peroxide can be reacted with toss (N-ethyl-N- (2-hydroxy-3-sulfopropyl) m-toluidine sodium salt) and 4-AP (4-aminoantipyrine) under the catalysis of horseradish peroxidase to produce a purple compound. Therefore, we assessed the change in activity of creatine amidinohydrolase by monitoring the amount of change in UV absorption of a single enzymatic reaction system at a wavelength of 555nm by a UV-2550 UV-visible spectrophotometer (Shimadzu), where unit activity is defined as the amount of enzyme producing 1. Mu.M hydrogen peroxide per minute.
The enzyme reaction system is as follows: 0.5mM TOOS (N-ethyl-N- (2-hydroxy-3-sulfopropyl) M-toluidine sodium salt), 0.45mM 4-AP (4-aminoantipyrine ), 900U/L horseradish peroxidase, 0.1M potassium phosphate buffer (pH 7.5).
1) The activity of creatine amidinohydrolase is measured by an enzyme multi-stage coupling method under the catalytic action of sarcosine oxidase and horseradish peroxidase, and a to-be-detected sample enzyme concentration is diluted to 1mg/ml by using a phosphate buffer solution (0.1M, pH 7.5). The substrate solution was prepared from 500. Mu.M creatine, 0.45mM 4-AA (4-aminoantipyrine), 0.5mM TOOS (N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline) and phosphate buffer (0.1M, pH 7.5), and incubated at 37 ℃. The enzyme activity was measured by taking 950. Mu.L of the substrate solution and adding 50. Mu.L of the enzyme sample to be tested thereto, and the change in the absorption of ultraviolet light at 555nm in the enzyme reaction system was monitored by a UV2550 spectrophotometer (Shimadzu), and the unit activity was defined as the amount of the enzyme that generates 1. Mu.M hydrogen peroxide per minute.
The creatine amidino hydrolase mutants provided by the invention comprise single-site mutants and combined mutants (shown in table 3), and the activity of wild-type creatine amidino hydrolase (BAA 88830.1) and creatine amidino hydrolase mutants at 57 ℃ is determined, so that compared with the optimized wild-type creatine amidino hydrolase (BAA 88830.1), the activity of four creatine amidino hydrolase mutants at 57 ℃ is obviously improved, and the four creatine amidino hydrolase mutants are respectively: K351E/L6P/T199S/T251C, K351E/L6P/F108Y/Y109F, Q165I/L6P/F108Y/Y109F/E349V, and K351E/L6P/F108Y/Y109F/Q165I, which are specifically shown in Table 3.
TABLE 3 wild-type creatine amidino hydrolase and its mutant activity
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence listing
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gaagaaatta aactgattcg cgaaggtgcc 30
<210> 14
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
cttctttaat ttgactaagc gcttccacgg 30
<210> 15
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
gcctctacca atgcaatgat tcgtgaaatt g 31
<210> 16
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
cggagatggt tacgttacta agcatttaac 30
<210> 17
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
gaattgcttt ccgatgattt ttggc 25
<210> 18
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
cttaacgaaa ggctactaaa aaccg 25
<210> 19
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
gataccgttc tgaaaccggg tatggttg 28
<210> 20
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
ctatggcaag actttggccc cataccaac 29
<210> 21
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
gataccgaac tggaaccggg tatggttgtt agtatg 36
<210> 22
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
catacccggt tccagttcgg tatcaatatc ttcgcg 36
Claims (6)
1. A creatine amidinohydrolase mutant, which is a combined mutant of any one combined mutation site in D17V/K351E, T199S/K351E, K351E/L6P/T199S/T251C, K351E/L6P/F108Y/Y109F/Q165I on the amino acid sequence of the GenBank accession number BAA 88830.1.
2. A gene encoding the creatine amidinohydrolase mutant according to claim 1.
3. A recombinant plasmid comprising the gene of claim 2.
4. An immobilized or engineered bacterium comprising the creatine amidinohydrolase mutant according to claim 1.
5. The engineered bacterium of claim 4, wherein said engineered bacterium comprises a fungal cell, a bacterial cell.
6. The engineered bacterium of claim 5, wherein the engineered bacterium comprises Escherichia coli, yeast, or Bacillus subtilis.
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| CN202211447024.2A CN115851681A (en) | 2020-08-28 | 2020-08-28 | Creatine amidino hydrolase mutant with improved activity |
| CN202010888788.XA CN112011529B (en) | 2020-08-28 | 2020-08-28 | Creatine amidino hydrolase mutant with improved activity |
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|---|---|---|---|---|
| CA2404293C (en) * | 2001-09-20 | 2007-05-15 | F. Hoffmann-La Roche Ag | Variants of an erwinia-type creatinase |
| CN105274080B (en) * | 2015-11-11 | 2018-08-07 | 江南大学 | A kind of creatine hydrolysis enzyme mutant that thermal stability improves |
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2020
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- 2020-08-28 CN CN202211447024.2A patent/CN115851681A/en active Pending
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| CN112011529A (en) | 2020-12-01 |
| CN115851681A (en) | 2023-03-28 |
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